Glycine-rich proteins as structural components of plant cell walls

Glycine-rich proteins (GRPs) have been found in the cell walls of many higher plants and form a third group of structural protein components of the wall in addition to extensins and proline-rich proteins. The primary sequences of GRPs contain more than 60% glycine. GRPs are localized mainly in the vascular tissue of the plant, and their coding genes provide an excellent system to analyze the molecular basis of vascular-specific gene expression. In French bean, the major cell wall GRP has been localized at the ultrastructural level in the modified primary cell wall of protoxylem. Immunological studies showed that it forms a major part of these highly extensible and specialized cell walls. Specific digestion of GRP1.8 from bean by collagenase suggests that it shares structural similarities with collagen. The protein is synthesized by living protoxylem cells as well as xylem parenchyma cells. After cell death, GRPs are exported from neighboring xylem parenchyma cells to the protoxylem wall, a rare example of protein transport between cells in plants. We propose that GRPs are part of a repair system of the plant during the stretching phase of protoxylem.     

In pre-sterol carrier protein 2 (SCP2) in solution the leader peptide 1 – 20 is flexibly disordered, and residues 21 – 143 adopt the same globular fold as in mature SCP2

The preform of the rabbit sterol carrier protein 2 (pre-rSCP2) was cloned, the uniformly ¹⁵N-labelled protein expressed in Escherichia coli and studied by three-dimensional ¹⁵N-resolved nuclear magnetic resonance spectroscopy. In spite of its low solubility in aqueous solution of only ∼0.3 mM, sequential ¹⁵N and ¹H backbone resonance assignments were obtained for 105 out of the 143 residues. From comparison of the sequential and medium-range nuclear Overhauser effects (NOEs) in the two proteins, all regular secondary structures previously determined in mature human SCP2 (hSCP2) [Szyperski et al. (1993) FEBS Lett. 335: 18–26] were also identified in pre-rSCP2. Near-identity of the backbone ¹⁵N and ¹H chemical shifts and 1 : 1 correspondence of 24 long-range NOEs to backbone amide groups in the two proteins show that the residues 21 – 143 adopt the same globular fold in pre-rSCP2 and mature hSCP2. The N-terminal 20-residue leader peptide of pre-rSCP2 is flexibly disordered in solution and does not observably affect the conformation of the polypeptide segment 21 – 143. Received 11 May 1998; accepted 15 May 1998     

BH3-only proteins in tumorigenesis and malignant melanoma

BH3-only proteins are a subset of the Bcl-2 family of apoptotic regulators. BH3-only proteins function as ‘damage sensors’ in the cell; they are activated in response to cellular stress or DNA damage, whereupon they initiate apoptosis. Apoptosis is the primary mechanism by which the body rids itself of genetically defective cells and is critical for preventing the accumulation of cells with tumorigenic potential. Therefore, dysregulation of BH3-only proteins may promote tumorigenesis. Furthermore, functional apoptosis pathways are required for the success of most cancer treatments, including chemotherapy. Resistance to chemotherapy, as seen with malignant melanoma, often reflects an inability of tumor cells to undergo apoptosis. By deciphering the roles of BH3-only proteins in tumorigenesis, we may learn how to manipulate cell death pathways to overcome apoptotic resistance. This review summarizes the current knowledge of BH3-only proteins and how they contribute to tumorigenesis, with particular attention given to studies involving melanoma. Received: 12 August 2006; received after revision: 2 October 2006; accepted 13 November 2006     

Assembly and genetics of spore protective structures

The sporulation program in Bacillus subtilis ends in the formation of a highly resistant endospore that can withstand extremes of heat, mechanical disruption, ultraviolet irradiation, lytic enzymes and chemical attack. These properties are attributed mainly to the unique structure of spore coat and cortex, as well as to the physical state of the spore cytoplasm. The outermost layer of the spore, called the coat, has two morphologically distinct sublayers: an electron-dense outer coat and an electron-translucent inner coat. The coat is composed of more than 2 dozen proteins of varying size. Many coat genes and coat proteins have been isolated and characterized in detail, and studies of these have identified proteins with important roles in coat assembly, resistance and spore germination. We describe here characteristics of the coat proteins and propose a model for coat assembly based on recent work.     

Signalling roles of mammalian phospholipase D1 and D2

Phospholipase D (PLD) catalyses the hydrolysis of phosphatidylcholine to generate the lipid second messenger, phosphatidate (PA) and choline. PLD activity in mammalian cells is low and is transiently stimulated upon activation by G-protein-coupled and receptor tyrosine kinase cell surface receptors. Two mammalian PLD enzymes (PLD1 and PLD2) have been cloned and their intracellular regulators identified as ARF and Rho proteins, protein kinase Cα as well as the lipid, phosphatidylinositol [4, 5] bisphosphate (PIP₂). I discuss the regulation of these enzymes by cell surface receptors, their cellular localisation and the potential function of PA as a second messenger. Evidence is presented for a role of PA in regulating the lipid kinase activity of PIP 5-kinase, an enzyme that synthesises PIP₂. A signalling role of phospholipase D via PA and indirectly via PIP₂ in regulating membrane traffic and actin dynamics is indicated by the available data. Received 25 April 2001; received after revision 15 June 2001; accepted 15 June 2001     

The structure and function of Escherichia coli penicillin-binding protein 3

Escherichia coli penicillin-binding protein PBP3 is a key element in cell septation. It is presumed to catalyse a transpeptidation reaction during biosynthesis of the septum peptidoglycan but, in vitro, its enzymatic activity has only been demonstrated with thiolester analogues of the natural peptide substrate. It has no detectable transglycosylase activity with lipid II as substrate. This tripartite protein is constructed of an N-terminal membrane anchor-containing module that is essential for cell septation, a non-penicillin-binding (n-PB) module of unknown function and a C-terminal penicillin-binding (PB) module exhibiting all the characteristic motifs of penicilloyl serine transferases. The n-PB module, which is required for the folding and stability of the PB module, may provide recognition sites for other cell division proteins. Initiation of septum formation is not PBP3-dependent but rests on the appearance of the FtsZ ring, and is thus penicillin-insensitive. The control of PBP3 activity during the cell cycle is briefly discussed.     

The mitochondrial PHB complex: roles in mitochondrial respiratory complex assembly, ageing and degenerative disease

Although originally identified as putative negative regulators of the cell cycle, recent studies have demonstrated that the PHB proteins act as a chaperone in the assembly of subunits of mitochondrial respiratory chain complexes. The two PHB proteins, Phb1p and Phb2p, are located in the mitochondrial inner membrane where they form a large complex that represents a novel type of membrane-bound chaperone. On the basis of its native molecular weight, the PHB-complex should contain 12-14 copies of both Phb1p and Phb2p. The PHB complex binds directly to newly synthesised mitochondrial translation products and stabilises them against degradation by membrane-bound metalloproteases belonging to the family of mitochondrial triple-A proteins. Sequence homology assigns Phb1p and Phb2p to a family of proteins which also contains stomatins, HflKC, flotillins and plant defence proteins. However, to date only the bacterial HflKC proteins have been shown to possess a direct functional homology with the PHB complex. Previously assigned actions of the PHB proteins, including roles in tumour suppression, cell cycle regulation, immunoglobulin M receptor binding and apoptosis seem unlikely in view of any hard evidence in their support. Nevertheless, because the proteins are probably indirectly involved in ageing and cancer, we assess their possible role in these processes. Finally, we suggest that the original name for these proteins, the prohibitins, should be amended to reflect their roles as proteins that hold badly formed subunits, thereby keeping the nomenclature already in use but altering its meaning to reflect their true function more accurately. Received 21 May 2001; received after revision 2 July 2001; accepted 24 July 2001     

Galileo’s quanti: understanding infinitesimal magnitudes

In On Local Motion in the Two New Sciences, Galileo distinguishes between ‘time’ and ‘quanto time’ to justify why a variation in speed has the same properties as an interval of time. In this essay, I trace the occurrences of the word quanto to define its role and specific meaning. The analysis shows that quanto is essential to Galileo’s mathematical study of infinitesimal quantities and that it is technically defined. In the light of this interpretation of the word quanto, Evangelista Torricelli’s theory of indivisibles can be regarded as a natural development of Galileo’s insights about infinitesimal magnitudes, transformed into a geometrical method for calculating the area of unlimited plane figures.     

Heat shock protein gene expression during Xenopus development

Stress-induced heat shock protein gene expression is developmentally regulated during early embryogen esis of the frog, Xenopus laevis. For example, a number of heat shock protein genes, such as hsp70, hsp90, and ubiquitin are not heat-inducible until after the midblastula stage of embryogenesis. Furthermore, the family of small heat shock protein genes, hsp30, are differentially expressed after the midblastula stage as well as being regulated at the level of mRNA stability. Many of these stress proteins are also synthesized constitutively during oogenesis and embryogenesis during which they may act as molecular chaperones as well as being involved in sequestering proteins in an inactive state until required by the developing embryo. Furthermore the induction of these stress protein genes has been correlated with enhanced thermoresistance. During stressful conditions heat shock proteins probably prevent aggregation or misfolding of damaged protei ns within the embryo.     

Annexins: what are they good for?

Annexins comprise a unique family of calcium- and phospholipid-binding proteins. At least one of the twenty members thus far described from this family can be found expressed in nearly every eukaryotic cell type. As common as these proteins may be, no one clear function for all has been established. Historically, individual members of this family have been given various names describing their ability to associate with a host of intra- and extracellular proteins and with cellular lipid membranes. The collection of reviews in this issue of CMLS represents an effort to offer a coordinated view of the research activities in the field and to extract structural and functional commonalities.     

Synthetic genes for the elucidation of glycosylation codes for arabinogalactan-proteins and other hydroxyproline-rich glycoproteins

Hydroxyproline-rich glycoproteins (HRGPs) are ubiquitous architectural components of the growing plant cell wall, accounting for as much as 10-20% of the dry weight. HRGPs are implicated in all aspects of plant growth and development, including responses to stress. The HRGP superfamily contains three major groups which represent a continuum of peptide periodicity and hydroxyproline-O-glycosylation. These groups range from the highly periodic and lightly arabinosylated repetitive proline-rich proteins (PRPs), through the crosslinked extensins which are periodic and highly arabinosylated, to the arabinogalactan-proteins (AGPs) which are the most highly glycosylated and least periodic. The repetitive units are small, often only four- to six-residue-glycosylated modules viewed hypothetically as functional motifs, or glycomodules. The Hyp contiguity hypothesis predicts that Hyp arabinosylation increases with Hyp contiguity and that clustered noncontiguous Hyp residues are sites of arabinogalactan polysaccharide addition in the AGPs and gums. Recent results involving glycosylation site mapping of endogenous HRGPs and HRGP design using synthetic genes have corroborated the hypothesis. The uses of synthetic genes in HRGP glycosylation site mapping and structural/functional analysis are also discussed.     

Occludin binds to the SH3-hinge-GuK unit of zonula occludens protein 1: potential mechanism of tight junction regulation

The interaction between tight junction proteins occludin and zona occludens protein 1 (ZO-1) was clarified. The sequence cc1 within the hinge region of ZO-1, connecting its SH3 and GuK domains, was identified as a new association site for the occludin C-terminus, core binding area GLRSSKRNLRKSR (mouse ZO-1^606-618). Occludin also bound to the sequence H2 within GuK, core area HKLRKNNH (ZO-1^759-766). In occludin, the binding core was ELSRLDKELDDYREESEEY (mouse occludin^455-473). Helicity of the sequences was suggested by circular dichroism. Because basic residues in ZO-1, acidic residues in occludin (underlined), coiled-coil helix-forming leucine heptad motifs (bold) in occludin and, probably, in cc1 were essential, we conclude that interactions were both helical and ionic. Moreover, the GuK domain bound other GuK molecules, suggesting oligomerization of ZO-1. Generally, the assumption is supported that the SH3-hinge-GuK region represents a functional and regulatory unit in ZO-1 forming a multiprotein tight junction complex with occludin.Received 12 January 2004; received after revision 23 February 2004; accepted 31 March 2004     

Novel regulation and function of Src tyrosine kinase

Src tyrosine kinase is a critical signal transducer that modulates a wide variety of cellular functions. Misregulation of Src leads to cell transformation and cancer. Heterotrimeric guanine-nucleotide-binding proteins (G proteins) are another group of signaling molecules that transduce signals from cell-surface receptors to generate physiological responses. Recently, it was discovered that Gαs and Gαi could directly stimulate Src family tyrosine kinase activity. This novel regulation of Src tyrosine kinase by G proteins provides insights into the adenylyl cyclase-independent signaling mechanisms involved in ligand-induced receptor desensitization, internalization and other physiological processes. Received 17 August 2001; received after revision 22 October 2001; accepted 24 October 2001     

Ras proteins in the control of the cell cycle and cell differentiation

The Ras family of small GTPases includes three closely related proteins: H-, K-, and N-Ras. Ras proteins are involved in the transduction of signals elicited by activated surface receptors, acting as key components by relaying signals downstream through diverse pathways. Mutant, constitutively activated forms of Ras proteins are frequently found in cancer. While constitutive Ras activation induces oncogenic-like transformation in immortalized fibroblasts, it causes growth arrest in primary vertebrate cells. Induction of p53 and cyclin-dependent kinase inhibitors such as p15^INK4b, p16^INK4a, p19^ARF, and p21^WAF1 accounts for this response. Interestingly, while ras has usually been regarded as a transforming oncogene, the analysis of Ras function in most of the cellular systems studied so far indicates that the promotion of differentiation is the most prominent effect of Ras. While in some cell types, particularly muscle, Ras inhibits differentiation, in others such as neuronal, adipocytic, or myeloid cells, Ras induces differentiation, in some cases accompanied by growth arrest. Several possible mechanisms for the pleiotropic effects of Ras in animal cells are discussed. Received 8 March 2000; received after revision 24 May 2000; accepted 24 May 2000     

G proteins as drug targets

The structure and function of heterotrimeric G protein subunits is known in considerable detail. Upon stimulation of a heptahelical receptor by the appropriate agonists, the cognate G proteins undergo a cycle of activation and deactivation; the α-subunits and the βγ-dimers interact sequentially with several reaction partners (receptor, guanine nucleotides and effectors as well as regulatory proteins) by exposing appropriate binding sites. For most of these domains, low molecular weight ligands have been identified that either activate or inhibit signal transduction. These ligands include short peptides derived from receptors, G protein subunits and effectors, mastoparan and related insect venoms, modified guanine nucleotides, suramin analogues and amphiphilic cations. Because compounds that act on G proteins may be endowed with new forms of selectivity, we propose that G protein subunits may therefore be considered as potential drug targets. Received 18 September 1998; received after revision 6 November 1998; accepted 11 November 1998     

Aberrant,canavanyl protein formation and the ability to tolerate or utilize L-canavanine

Summary L-Canavanine, 2-amino-4-(guanidinooxy)butyric acid, and L-arginine incorporation into de novo synthesized proteins was compared in six organisms. Utilizing L-[guanidinooxy¹⁴C]canavanine and L-[guanidino¹⁴C]arginine at substrate saturation, the canavanine to arginine incorporation ratio was determined in de, novo synthesized proteins.Caryedes brasiliensis andSternechus tuberculatus, canavanine utilizing insects;Canavalia ensiformis, a canavanine storing plant; and to a lesser extentHeliothis virescens, a canavanine resistant insect, failed to accumulate significant canavanyl proteins. By contrast,Manduca sexta, a canavanine-sensitive insect, andGlycine max, a canavanine free plant, readily incorporated canavanine into newly synthesized proteins. This study supports the contention that the incorporation of canavanine into proteins in place of arginine contributes significantly to canavanine's antimetabolic properties.     

Evidence of undiscovered cell regulatory mechanisms: phosphoproteins and protein kinases in mitochondria

The finding that mitochondria contain substrates for protein kinases lead to the discovery that protein kinases are located in the mitochondria of certain tissues and species. These include pyruvate dyhydrogenase kinase, branched-chain α-ketoacid dehydrogenase kinase, protein kinase A, protein kinase Cδ, stress-activated kinase and A-Raf as well as unidentified kinases. Recent evidence suggests that mitochondrial protein kinases may be involved in physiological processes such as apoptosis and steroidogenesis. Additionally, the novel finding of low-molecular-weight GTP-binding proteins in mitochondria suggests the possibility that these may interact with mitochondrial protein kinases to regulate the activity of mitochondrial effector proteins. The fact that there are components of cellular regulatory systems in mitochondria indicates the exciting possibility of undiscovered systems regulating mitochondrial physiology. Received 19 June 2001; received after revision 7 August 2001; accepted 8 August 2001     

The clostridial mobilisable transposons

Mobilisable transposons are transposable genetic elements that also encode mobilisation functions but are not in themselves conjugative. They rely on coresident conjugative elements to facilitate their transfer to recipient cells. Clostridial mobilisable transposons include Tn4451 and Tn4452 from Clostridium perfringens, and Tn4453a and Tn4453b from Clostridium difficile, all of which are closely related, and Tn5398 from C. difficile. The Tn4451 group of elements encodes resistance to chloramphenicol and is unusual in that transposition is dependent upon a large resolvase protein rather than a more conventional transposase or integrase. This group of elements also encodes the mobilisation protein TnpZ that, by acting at the RS_A or oriT site located on the transposon, and in the presence of a coresident conjugative element, promotes the movement of the nonreplicating circular intermediate and of plasmids on which the transposon resides. The erythromycin resistance element Tn5398 is unique in that it encodes no readily identifiable transposition or mobilisation proteins. However, the element is still capable of intraspecific transfer between C. difficile isolates, by an unknown mechanism. The detailed analysis of these mobilisable clostridial elements provides evidence that the evolution and dissemination of antibiotic resistance genes is a complex process that may involve the interaction of genetic elements with very different properties. RID="*" ID="*"Corresponding author.     

Antifungal proteins: targets,mechanisms and prospective applications

All organisms have evolved several defence systems in order to protect themselves against bacteria, fungi and viruses. Higher organisms have developed a complex network of humoral and cellular responses, called adaptive immunity. A second defence system, innate immunity, was discovered in the early 1980s, consisting of small cationic peptides with a broad antimicrobial spectrum. These proteins act immediately at sites of infection or inflammation. The production of proteins with antimicrobial activity was not limited to higher organisms but was also found in insects, plants and microorganisms. During the last 2decades a broad range of proteins with very different structural features have been isolated and characterised from differing organisms ranging from bacteria to human beings. Over 500cationic membrane-acting proteins with antimicrobial and antifungal activities have been identified to date. Apart from these proteins, a very large number of antifungal proteins active on the fungal cell wall, on enzymes of the cell wall synthesis machinery, the plasma membrane and on intracellular targets have been characterised.Received 17 June 2003; received after revision 4 August 2003; accepted 18 August 2003     

Protein localization in the plant Golgi apparatus and the <Emphasis Type="Italic">trans</Emphasis>-Golgi network

This review presents plant-specific characteristics of the Golgi apparatus and discusses their impact on retention of membrane proteins in the Golgi or the trans-Golgi network (TGN). The plant Golgi consists of distinct stacks of cisternae that actively move throughout the cytoplasm. The Golgi apparatus is a very dynamic compartment and the site for maturation of N-linked glycans. It is also a factory for complex carbohydrates that are part of the cell wall. The TGN is believed to be the site from where vacuolar proteins are sorted by receptors towards each type of vacuole. To maintain the structure and specific features of the Golgi, resident proteins ought to be maintained in the proper Golgi cisternae or in the TGN. Two families of membrane proteins will be taken as examples for Golgi/TGN retention: (i) the enzymes involved in N-glycosylation processes and (ii) a vacuolar sorting receptor. Although the number of available plant proteins localized in Golgi/TGN is low, the basis of retention appears to be shared over all kingdoms and may result from pure retention and recycling mechanisms. In this review, we will summarize the characteristics of a plant Golgi and will discuss especially their consequences on on the study of this highly dynamic structure. We then choose membrane proteins with a single transmembrane domain to illustrate the signals and mechanisms involved in plants to localize and maintain proteins in the Golgi and the TGN.